Congenital adrenal hyperplasia (CAH) is an inherited adrenal steroid synthesis disorder, included in the Dutch Newborn Screening (NBS) since 2002. The NBS for CAH consists of a 17-hydroxyprogesterone (17-OHP) measurement in dried blood spots (DBS) with gestational age–adjusted cutoffs. Until October 2021, a second heel prick (HP) was performed in newborns with inconclusive results to reduce false-positives. Since then, a 21-deoxycortisol (21-DOCL) measurement in the first DBS was introduced as a second tier if results were inconclusive to replace the second HP. The aim of the present study is to evaluate the performance of the second tier between 1 October 2021 and 31 December 2022. 

DBS of newborns with inconclusive and positive 17-OHP screening results, measured by immunoassay, were sent from regional NBS laboratories to the Amsterdam UMC Endocrine Laboratory. 21-DOCL and 17-OHP concentrations were analyzed by LC-MS/MS. Diagnoses were confirmed by mutation analysis.

Over a period of 15 months, 21-DOCL was measured in DBS of 93 newborns which is 0.04% of all screened newborns (215.196). Of these 93 newborns, nine were directly referred based on the positive 17-OHP result, of which seven also had a positive 21-DOCL and were diagnosed with CAH. 84 newborns had an inconclusive 17-OHP result, and only one of them, a premature newborn, had a positive 21-DOCL and was referred but did not have CAH. In total, ten newborns were referred of which seven were diagnosed with CAH resulting in three false-positive referrals; two based on the positive 17-OHP result and one on an inconclusive 17-OHP result and a positive second tier. The other 83 newborns did not have CAH. One false-negative result was reported based on a 17-OHP result below the cut-off value.

The modified protocol improves the CAH screening by preventing 84 second heel pricks in 15 months and also reducing the number of unnecessary referrals. Further research is needed to elucidate the reason for a false-positive second tier result as 21-DOCL is seen as a highly specific marker for CAH.